http://life-innov.phar.kyushu-u.ac.jp/Work in my laboratory is primarily directed to elucidating glia-neuron interactions in the spinal cord and brain and to understanding the cellular and molecular mechanisms of pain and itch signaling (in particular pathological chronic pain and itch). Our work employs several cutting-edge genetic tools to manipulate the specific type of cells to determine how identifiable populations of neurons and glial cells contribute to pain and itch behaviors in a causal manner. We also complementally use electrophysiology and cellular functional imaging (in vitro and in vivo) to measure activity of neurons and glial cells. From these studies, we devise strategies for new types of pain and itch relieving medications. .

Tsuda M, Tozaki-Saitoh H, Inoue K., Purinergic system, microglia and neuropathic pain., Current opinion in Pharmacology, 2012.02, Extracellular nucleotides play pivotal roles in the regulation of neuronal and glial functions in the nervous system through P2X receptors (P2XRs) and P2Y receptors (P2YRs). A growing body of evidence shows that microglia express several subtypes of P2XRs and P2YRs, and that these receptors play a key role in pain signaling in the spinal cord under pathological conditions, such as following peripheral nerve injury (neuropathic pain). Following peripheral nerve injury, dorsal horn microglia become activated and show upregulated expression of purinergic receptors, and interference with the function or expression of these receptors strongly suppresses neuropathic pain. This article highlights recent advances that further increase our understanding of the mechanisms by which microglial purinergic receptors contribute to the pathogenesis of neuropathic pain..

Hidetoshi Saitoh, Junya Masuda, Ryu Kawada, Chinami Kojima, Sosuke Yoneda, Takahiro Masuda, Kazuhide Inoue, Tsuda Makoto, Transcription factor MafB contributes to the activation of spinal microglia underlying neuropathic pain development, GLIA, 10.1002/glia.23570, 67, 4, 729-740, 2019.04, Microglia, which are pathological effectors and amplifiers in the central nervous system, undergo various forms of activation. A well-studied microglial-induced pathological paradigm, spinal microglial activation following peripheral nerve injury (PNI), is a key event for the development of neuropathic pain but the transcription factors contributing to microglial activation are less understood. Herein, we demonstrate that MafB, a dominant transcriptional regulator of mature microglia, is involved in the pathology of a mouse model of neuropathic pain. PNI caused a rapid and marked increase of MafB expression selectively in spinal microglia but not in neurons. We also found that the microRNA mir-152 in the spinal cord which targets MafB expression decreased after PNI, and intrathecal administration of mir-152 mimic suppressed the development of neuropathic pain. Reduced MafB expression using heterozygous Mafb deficient mice and by intrathecal administration of siRNA alleviated the development of PNI-induced mechanical hypersensitivity. Furthermore, we found that intrathecal transfer of Mafb deficient microglia did not induce mechanical hypersensitivity and that conditional Mafb knockout mice did not develop neuropathic pain after PNI. We propose that MafB is a key mediator of the PNI-induced phenotypic alteration of spinal microglia and neuropathic pain development..

Uesugi A, Kataoka A, Tozaki-Saitoh H, Koga Y, Tsuda M, Robaye B, Boeynaems JM, Inoue K., Involvement of protein kinase D in uridine diphosphate-induced microglial macropinocytosis and phagocytosis., Glia, 60, 7, 1094-105, 2012.07, The clearance of tissue debris by microglia is a crucial component of maintaining brain homeostasis. Microglia continuously survey the brain parenchyma and utilize extracellular nucleotides to trigger the initiation of their dynamic responses. Extracellular uridine diphosphate (UDP), which leaks or is released from damaged neurons, has been reported to stimulate the phagocytotic activity of microglia through P2Y(6) receptor activation. However, the intracellular mechanisms underlying microglial P2Y(6) receptor signals have not been identified. In this study, we demonstrated that UDP stimulation induced immediate and long-lasting dynamic movements in the cell membrane. After 60 min of UDP stimulation, there was an upregulation in the number of large vacuoles formed in the cell that incorporate extracellular fluorescent-labeled dextran, which indicates microglial macropinocytosis. In addition, UDP-induced vacuole formation and continuous membrane motility were suppressed by the protein kinase D (PKD) inhibitors, Gö6976 and CID755673, unlike Gö6983, which is far less sensitive to PKD. The inhibition of PKD also reduced UDP-induced incorporation of fluorescent-labeled dextran and soluble β-amyloid and phagocytosis of microspheres. UDP induced rapid phosphorylation and membrane translocation of PKD, which was abrogated by the inhibition of protein kinase C (PKC) with Gö6983. However, Gö6983 failed to suppress UDP-induced incorporation of microspheres. Finally, we found that inhibition of PKD by CID755673 significantly suppressed UDP-induced engulfment of IgG-opsonized microspheres. These data suggest that a PKC-independent function of PKD regulates UDP-induced membrane movement and contributes to the increased uptake of extracellular fluid and microspheres in microglia..

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Toyomitsu E, Tsuda M, Yamashita T, Tozaki-Saitoh H, Tanaka Y, Inoue K., CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia., Purinergic Signaling, 8, 2, 301-10, 2012.06, P2X4 receptors (P2X4Rs), a subtype of the purinergic P2X family, play important roles in regulating neuronal and glial functions in the nervous system. We have previously shown that the expression of P2X4Rs is upregulated in activated microglia after peripheral nerve injury and that activation of the receptors by extracellular ATP is crucial for maintaining nerve injury-induced pain hypersensitivity. However, the regulation of P2X4R expression on the cell surface of microglia is poorly understood. Here, we identify the CC chemokine receptor CCR2 as a regulator of P2X4R trafficking to the cell surface of microglia. In a quantitative cell surface biotinylation assay, we found that applying CCL2 or CCL12, endogenous ligands for CCR2, to primary cultured microglial cells, increased the levels of P2X4R protein on the cell surface without changing total cellular expression. This effect of CCL2 was prevented by an antagonist of CCR2. Time-lapse imaging of green fluorescent protein (GFP)-tagged P2X4R in living microglial cells showed that CCL2 stimulation increased the movement of P2X4R-GFP particles. The subcellular localization of P2X4R immunofluorescence was restricted to lysosomes around the perinuclear region. Notably, CCL2 changed the distribution of lysosomes with P2X4R immunofluorescence within microglial cells and induced release of the lysosomal enzyme β-hexosaminidase, indicating lysosomal exocytosis. Moreover, CCL2-stimulated microglia enhanced Akt phosphorylation by ATP applied extracellularly, a P2X4R-mediated response. These results indicate that CCL2 promotes expression of P2X4R protein on the cell surface of microglia through exocytosis of P2X4R-containing lysosomes, which may be a possible mechanism for pain hypersensitivity after nerve injury..

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Tsuda M, Tozaki-Saitoh H, Inoue K., Platelet-activating factor and pain., Biological and pharmaceutical bulletin, 34, 8, 1159-62, 2012.06, Platelet-activating factor (PAF) is a phospholipid mediator that regulates the functions of a variety of cells in the peripheral tissues and in the nervous system. Findings that injection of PAF exogenously at the skin or in the spinal cord induced pain hypersensitivity gave us much attention to its role in pain signaling. Studies using pharmacological and genetic tools to control the functions of the PAF receptor (PAFR) revealed that the PAF/PAFR system plays a role in tissue injury-induced pain, but not in the acute physiological pain evoked by thermal and mechanical stimuli. Recent investigations have focused on the roles of PAFR in pathological chronic pain such as the neuropathic pain that occurs after nerve injury for which there is currently no effective therapy. Nerve injury upregulated PAFRs in dorsal root ganglion (DRG) neurons. Studies using PAFR antagonists and PAFR-deficient mice indicated a crucial role of PAFR in production of tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) in the DRG and in developing and maintaining neuropathic pain. Thus, blocking PAFRs may be a viable therapeutic strategy for treating neuropathic pain..

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Kataoka A, Koga Y, Uesugi A, Tozaki-Saitoh H, Tsuda M, Inoue K., Involvement of vasodilator-stimulated phosphoprotein in UDP-induced microglial actin aggregation via PKC- and Rho-dependent pathways., Purinergic Signaling, 7, 4, 403-11, 2011.12, Microglia are major immunocompetent cells in the central nervous system and retain highly dynamic motility. The processes which allow these cells to move, such as chemotaxis and phagocytosis, are considered part of their functions and are closely related to purinergic signaling. Previously, we reported that the activation of the P2Y(6) receptor by UDP stimulation in microglia evoked dynamic cell motility which enhanced their phagocytic capacity, as reported by Koizumi et al. (Nature 446(7139):1091-1095, 2007). These responses require actin cytoskeletal rearrangement, which is seen after UDP stimulation. However, the intracellular signaling pathway has not been defined. In this study, we found that UDP in rat primary microglia rapidly induced the transient phosphorylation at Ser157 of vasodilator-stimulated phosphoprotein (VASP). VASP, one of actin binding protein, accumulated at the plasma membrane where filamentous (F)-actin aggregated in a time-dependent manner. The phosphorylation of VASP was suppressed by inhibition of PKC. UDP-induced local actin aggregations were also abrogated by PKC inhibitors. The Rho inhibitor CT04 and the expression of p115-RGS, which suppresses G(12/13) signaling, attenuated UDP-induced phosphorylation of VASP and actin aggregation. These results indicate that PKC- and Rho-dependent phosphorylation of VASP is involved in UDP-induced actin aggregation of microglia..

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Tozaki-Saitoh H, Tsuda M, Inoue K., Role of purinergic receptors in CNS function and neuroprotection., Advances in pharmacology , 61, 495-528, 2011.10, The purinergic receptor family contains some of the most abundant receptors in living organisms. A growing body of evidence indicates that extracellular nucleotides play important roles in the regulation of neuronal and glial functions in the nervous system through purinergic receptors. Nucleotides are released from or leaked through nonexcitable cells and neurons during normal physiological and pathophysiological conditions. Ionotropic P2X and metabotropic P2Y purinergic receptors are expressed in the central nervous system (CNS), participate in the synaptic processes, and mediate intercellular communications between neuron and gila and between glia and other glia. Glial cells in the CNS are classified into astrocytes, oligodendrocytes, and microglia. Astrocytes express many types of purinergic receptors, which are integral to their activation. Astrocytes release adenosine triphosphate (ATP) as a "gliotransmitter" that allows communication with neurons, the vascular walls of capillaries, oligodendrocytes, and microglia. Oligodendrocytes are myelin-forming cells that construct insulating layers of myelin sheets around axons, and using purinergic receptor signaling for their development and for myelination. Microglia also express many types of purinergic receptors and are known to function as immunocompetent cells in the CNS. ATP and other nucleotides work as "warning molecules" especially by activating microglia in pathophysiological conditions. Studies on purinergic signaling could facilitate the development of novel therapeutic strategies for disorder of the CNS..

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Kuboyama K, Harada H, Tozaki-Saitoh H, Tsuda M, Ushijima K, Inoue K., Astrocytic P2Y(1) receptor is involved in the regulation of cytokine/chemokine transcription and cerebral damage in a rat model of cerebral ischemia., Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism., 10.1038/jcbfm.2011.49, 31, 9, 1930-41, 2011.09, After brain ischemia, significant amounts of adenosine 5'-triphosphate are released or leaked from damaged cells, thus activating purinergic receptors in the central nervous system. A number of P2X/P2Y receptors have been implicated in ischemic conditions, but to date the P2Y(1) receptor (P2Y(1)R) has not been implicated in cerebral ischemia. In this study, we found that the astrocytic P2Y(1)R, via phosphorylated-RelA (p-RelA), has a negative effect during cerebral ischemia/reperfusion. Intracerebroventricular administration of the P2Y(1)R agonist, MRS 2365, led to an increase in cerebral infarct volume 72 hours after transient middle cerebral artery occlusion (tMCAO). Administration of the P2Y(1)R antagonist, MRS 2179, significantly decreased infarct volume and led to recovered motor coordination. The effects of MRS 2179 occurred within 24 hours of tMCAO, and also markedly reduced the expression of p-RelA and interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein-1/chemokine (C-C motif) ligand 2 (CCL2), and interferon-inducible protein-10/chemokine (C-X-C motif) ligand 10 (CXCL10) mRNA. P2Y(1)R and p-RelA were colocalized in glial fibrillary acidic protein-positive astrocytes, and an increase in infarct volume after MRS 2365 treatment was inhibited by the nuclear factor (NF)-κB inhibitor ammonium pyrrolidine dithiocarbamate. These results provide evidence that the P2Y(1)R expressed in cortical astrocytes may help regulate the cytokine/chemokine response after tMCAO/reperfusion through a p-RelA-mediated NF-κB pathway.

Chronic intractable pain, such as tactile allodynia, is recognized as the first subject to clinical treatment. There are accumulating evidences that several molecules related to activated microglia in the spinal cord play significant roles in development of allodynia. We have previously reported that P2X4 receptors are one of the key molecules for emerging allodynia. However, little is known how their expression is regulated. Recently, the modulation of retinoid signaling in the site of peripheral nerve injury was reported. In this study, we assessed the roles of retinoid signaling in the experimental allodynia. Oral administration of all-trans retinoic acid enhanced the development of tactile allodynia, which was accompanied by increase of P2X4 receptor expression in the spinal cord. The effect of retinoic acid was diminished by a retinoid X receptor pan-antagonist. Furthermore, the antagonist reversed pain hyper-sensitization and decreased spinal P2X4 receptor expression. We found that retinoids stimulate P2X4 receptor expression in primary microglia. Our findings suggest that retinoid signaling is the key pathway in the development of allodynia via the enhancement of microglial P2X4 receptor expression., [URL].

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Extracellular ATP is an important signaling molecule in the central nervous systems and is known as an activator of microglia. ATP-stimulated microglia dramatically changes their properties including morphology and produce and release cytokines and growth factors. Recent studies have shown that reactive microglia have roles in the pathological states as a consequence of neural damages. We have previously shown that P2X4 receptor, one of ATP-gated ionotropic receptors, expressed in spinal cord microglia is exclusively increased after peripheral nerve injury, and that activation of P2X4 receptors are required for neuropathic pain. The upregulation of P2X4 receptor in microglia is therefore a key process in producing neuropathic pain, but the mechanisms remain unknown. In this study, we first predicted a putative retinoic acid response element (RARE) in the 5’-flanking region of rat P2X4 gene (P2rx4) using a web-based homology search system. The luciferase reporter gene assay revealed the presence of a site sensitive to retinoic acid in the 5’-flanking region of P2rx4 we have cloned. In the rat primary cultured microglia, treatment with 9-cis retinoic acid (9cisRA) significantly increased both mRNA and protein levels of the P2X4 receptor. Synthetic agonists for retinoic acid receptor (RAR) and retinoid X receptor (RXR) synergistically increased the level of P2X4 mRNA, and pretreatment with an RXR antagonist inhibited the increase in the P2X4 mRNA by 9cisRA, indicating an involvement of RXR/RAR heterodimers. In in vivo studies, oral administration of all-trans retinoic acid (atRA) to rats enhanced both the development of tactile allodynia, and upregulation of P2X4 in the ipsilateral spinal cord after nerve injury. Our findings suggest that RXR and/or RAR, the nuclear receptors for retinoids, might stimulate the transcription of microglial P2X4 receptors and lead to the pain hypersensitivity after the nerve injury., [URL].

Membership in Academic Society

The Japan Neuroscience Society

The Japanese Pharmacological Society

Educational

Educational Activities

In order to achive voluntary theory establishment of students, my educational acivities are focused on teaching basic methodologies and multidimensional approaches for prove their hypothesis.